Rewiring of primary metabolism for ammonium recycling under short-term low CO2 treatment – its implication for C4 evolution

The dramatic decrease in atmospheric CO2 concentration during Oligocene was proposed as directly linked to C4 evolution. However, it remains unclear how the decreased CO2 concentration directly facilitate C4 evolution, besides its role as a selection pressure. We conducted a systematic transcriptomics and metabolomics analysis under short-term low CO2 condition and found that Arabidopsis grown under this condition showed 1) increased expression of most genes encoding C4-related enzymes and transporters; 2) increased expression of genes involved in photorespiration and pathways related to carbon skeleton generation for ammonium refixation; 3) increased expression of genes directly involved in ammonium refixation. Furthermore, we found that in vitro treatment of leaves with NH4 + induced a similar pattern of changes in C4 related genes and genes involved in ammonium refixation. These data support the view that Arabidopsis grown under short-term low CO2 conditions rewired its metabolism to supply carbon skeleton for ammonium recycling, during which process the expression of C4 genes were up-regulated as a result of a hitchhiking process. This study provides new insights into the adaptation of the C3 model plant Arabidopsis under low CO2 conditions and suggests that low CO2 can facilitate the evolution of C4 photosynthesis beyond the commonly assumed role of being a selection pressure.


Introduction
Although C 4 plants only have 8000 species, but it contributes to 23% of the earth's terrestrial primary productivity (Still et al., 2003;Sage et al., 2007).With over 60 distinct evolutionary origins (Sage et al., 2011;Sage et al, 2018), C 4 photosynthesis represents an excellent example of convergent evolution, which attracts attentions among biologists to study the evolutionary history of C 4 photosynthesis (Sage, 2001;Sage et al., 2011).According to the present models of C 4 evolution (Sage, 2011;Sage, 2012), relocation of photorespiratory CO 2 release to the bundle sheath cell (BSC) from the mesophyll cell (MC) is an important early step during the evolution of the C 4 photosynthesis (Vogan et al., 2007;Sage, 2012).This step results in C 2 photosynthesis, which is characterized by a photorespiratory CO 2 pump can elevate the concentration of CO 2 in BSC by ~threefold (Keerberg et al., 2014).Immunogold experiments show that glycine decarboxylase (GDC) is expressed exclusively in the BSC of some intermediates, such as Moricandia arvensis (Rawsthorne et al., 1988;Schluter et al., 2017) and Heliotropium (Vogan et al., 2007).Due to the enrichment of CO 2 , Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) works more efficiently in the BSC, leading to an overall increase in biomass production under condition which supports higher rates of photorespiration (Heckmann et al., 2013;Mallmann et al., 2014), such as low CO 2 , drought and high temperature (Wingler et al., 1999;Carmo-Silva et al., 2008;Li Y. et al., 2014;Sage et al., 2018).
Accompanied with the release of CO 2 by GDC during photorespiration, ammonium (NH 4 + ) is also released (Bauwe et al., 2010), which needs to be refixed since it is toxic (Krogmann et al., 1959).Photorespiratory ammonium can be refixed and integrated into amino acids via the chloroplastic isoform Glutamine synthetase 2 (GS2) and ferredoxin-dependent GOGAT (Fd-GOGAT) (Lam et al., 1996).This process forms the photorespiratory ammonium cycle (Keys, 2006).Photorespiratory CO 2 pump results in ammonium imbalance between MC and BSC, which needs to be solved through metabolic shuttles (Nagatani et al., 1971;Lancien et al., 2000;Liu and von Wiren, 2017).Mallman (Mallmann et al., 2014) showed that there are multiple solutions to the ammonium imbalance, with one option, an alanine-pyruvate shuttle, providing a bridge toward C 4 photosynthesis.Therefore, ammonium refixation induced by high rate of photorespiration was proposed a bridge toward C 4 evolution.However, it raises a question regarding how these metabolic shuttles which recycle ammonium emerge during C 4 evolution.Considering that these metabolic shuttles for ammonium recycling require a rather large scale rewiring of the plant's primary metabolism, quickly evolving such a mechanism when ammonium imbalance in C 2 plants appeared would be rather diffi cult.Importantly, C 4 photosynthesis mostly emerged around 30 million years ago and originated independently more than 60 times (Sage et al., 2011), there seems little likelihood for such complex metabolic pathways and its associated regulatory mechanisms to evolve multiple times in such a short time.All genes involved in C 4 photosynthesis are present in C 3 ancestral plants and play important house-keeping roles (Aubry et al., 2011); similarly, all the genes involved in the ammonium recycling already exist in gymnosperm species (Valderrama-Martıń et al., 2022).It however remains to be tested whether there are regulatory mechanisms coordinating the expression of these genes under conditions favoring photorespiration in typical C 3 plants.
The 30-million-year time span subsequent to the Oligocene CO 2 decrease during which numerous C 4 plants originated suggests that historically low CO 2 levels most likely facilitated C 4 evolution (Ehleringer et al., 1991;Ehleringer et al, 1997;Christin et al., 2008).Low CO 2 condition enhances photorespiration (Li Y. et al., 2014).However, there is little study so far on how this low CO 2 directly contributes to the evolutionary formation of the C 4 pathway genetically.In this study, we attempt to answer this question through combined transcriptomics and metabolomics analysis.Given that under short-term low CO 2 treatment, changes in enzymatic sequences would not be expected, this study focuses on test whether low CO 2 altered expressions of genes related to C 4 photosynthesis and how.Our results suggest that low CO 2 condition indeed induced metabolic rewiring to increase ammonia refixation in the C 3 model plant Arabidopsis thaliana, though the signaling pathways through which low CO 2 induced observed changed gene expression remains unknown.Since many reactions involved in the ammonia refixation overlap with those for C 4 photosynthesis, the mechanisms responsible for the upregulation of genes related to ammonia refixation inevitably facilitated the up-regulation of genes related to C 4 photosynthesis.Therefore, the up-regulation of C 4 photosynthesis represents an excellent example of hitchhiking process for the emergence of an important biological process.

Plant material and growth conditions
Arabidopsis used in this study is the Columbia (Col-0) ecotype.Seeds were surface-sterilized by incubation with 10% (v/v) bleach diluted with ethanol, washed three times with sterile distilled water, and then sown in plastic Petri dishes (diameter 90 mm, depth 20 mm) containing 1/2 MS medium, solidified with 0.7% (w/v) agar and supplemented with 1.5% (w/v) sucrose (pH 5.8).The seeds were placed in the dark at 4°C for three days and germinated for six days in incubators with a photosynthetic photon flux density (PPFD) of 100 mmol m -2 s -1 (10 h light/14 h dark cycle) at 22°C.After this, seedlings were transferred to Pindstrup soil and grown in a Percival incubator (Nihonika, Japan) in which CO 2 gas was accurately and stably controlled.

Low CO 2 treatment
The CO 2 condition of 100 ppm (low CO 2 , LC) and 400 ppm (normal CO 2 , NC) were applied in two separate Percival incubators (Nihonika, Japan) and maintained throughout this study.Arabidopsis were grown for 14 days under a PPFD of 100 mmol m -2 s -1 (10 h light/14 h dark cycle) at 22°C and relative humidity of 65% (Li Y. et al., 2014).After, half of the plants were transferred to low CO 2 condition for six days.New fully expanded leaves in threeweek-old plants were sampled at 11 am in the morning for transcriptomic and metabolomics analysis, the measurement of chlorophyll content and free ammonium levels under both low CO 2 and normal CO 2 levels.

Ammonium treatment
To study the effect of ammonium ions on gene expression, seeds were surface-sterilized and sown in a control check (CK) plastic Petri dish (diameter 90 mm, depth 20 mm) in a growth chamber for 10 days at a PPFD of 100 mmol m -2 s -1 (10 h light/14 h dark cycle) at 22°C . The composition of the CK was described in (Li et al., 2013), which is composed of 2 mM KH 2 PO 4 , 5 mM NaNO 3 , 2 mM MgSO 4 , 1 mM CaCl 2 , 0.1 mM Fe-EDTA, 50 mM H 3 BO 3 , 12 mM MnSO 4 , 1 mM ZnCl 2 , 1 mM CuSO 4 , 0.2 mM Na 2 MoO 4 , 0.5 gl -1 MES, 1% sucrose, and 0.8% agarose (pH 5.7, adjusted with 1 M NaOH).Half of the seedlings were transferred to the new CK petri dish and the remaining seedlings were transferred to new CK plates which were additionally supplied with 15 mM or 30 mM (NH 4 ) 2 SO 4 .Nine seedlings were put on one Petri dish and considered as one biological replicate during sampling.After three days of treatments, leaves were harvested and immediately put into liquid nitrogen and stored at -80°C until RNA extraction.We also put seedlings on the CK plates which were additionally supplied with 30 mM or 60 mM NH 4 Cl, 15 mM or 30 mM K 2 SO 4 , 30 mM or 60 mM KNO 3 , and 120 mM mannitol to test the effect of other ions (Li et al., 2019).

Measurement of free ammonium content in a leaf
Ammonium content was determined with the colorimetric method based on the phenol hypochlorite assay (Soloŕzano, 1969;Sarasketa et al., 2014).Briefly, leaves were collected, weighed, and snap-frozen in liquid nitrogen, and then ground with 5 mm zirconia beads. 1 mL H 2 O was added to the frozen powder.The mixtures were incubated at 80°C for 10 min and then centrifuged at 4000 g and 4°C for 20 min.100 μL of supernatant was mixed with 200 mL of 0.33 M sodium phenolate, 100 μL of 0.02% sodium nitroprusside; then 200 μL of 2% sodium hypochlorite were added to the mixture.The mixture was incubated at room temperature for 30 min followed by reading absorbance at a wavelength of 630 nm in a spectrophotometer.

Gene expression analysis
For gene expression profiling, we took illuminated new fully expanded leaf samples.Four biological replicates were harvested and frozen in liquid nitrogen immediately and stored at -80°C before RNA extraction.For RNA extraction, the PureLink RNA Mini Kit (Life Technologies Corporation, USA) was used to extract RNA following the manufacturer's protocol, which includes the removal of gDNA from RNA.The quality of purified RNA was assessed using an Agilent 2100 Bioanalyzer (Agilent, USA) and the RNA samples with RNA Integrity Number (RIN) higher than 7 were used for RNA library construction.RNA libraries were prepared based on standard Illumina (Illumina, Inc., USA) protocols and sequenced using the Illumina X Ten platform in paired-end 150 bp mode.The quality of RNA-seq data (fastq files) was assessed by the FastQC software (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/).RNAseq analysis was performed by the STAR software (Dobin et al., 2013) with the Arabidopsis reference genome as well as a gene transfer format (GTF) file (downloaded from EnsemblPlants http:// plants.ensembl.org/).After generation of the genome index, RNAseq reads were aligned by STAR with the '-quantMode GeneCounts' option to count reads per gene.Differentially expressed genes (DEGs) were determined by the R package 'DESeq2' (Love et al., 2014) which use the Benjamini-Hochberg (BH) adjustment for multiple testing problem with the read counts reported by STAR (Dobin et al., 2013).DEGs between treatment and control were analyzed.Only genes with the adjusted P-value <0.05 (Padj) were considered as DEGs in each treatment.

Functional analysis of differentially expressed genes
Gene transcript abundances and metabolite abundances were visualized applying R package 'heatmap' (https://cran.r-project.org/web/packages/pheatmap/index.html).Permutation of Pearson correlations was conducted used the R package Permutation test.Volcano plot and Venn diagrams were plotted applying online tool Bioinformatics (https://www.bioinformatics.com.cn).To identify functional categories of differentially expressed genes, Gene Oncology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) (https://david.ncifcrf.gov/).Data were analyzed using the two-tailed Student's t-test.The term significant is used here for differences or correlations confirmed at * P < 0.05 or better.Details are provided in the figure legends.

Liquid chromatography/mass spectrometry and metabolomics analysis
To enable the sampling of leaves grown under low CO 2 , we modified the glass door of a Percival incubator to ensure the stability of CO 2 condition during leaf sampling.Specifically, we cut two spherical openings in the middle of the glass door and installed two plastic gloves used for sampling.
The Liquid Chromatography/Mass Spectrometry (LC-MS/MS) experiments were performed following (Wang et al., 2014;Arrivault et al., 2019).Fully expanded leaves under light were sampled.For each sample, a leaf area of 1.7 cm 2 was sampled and frozen in liquid nitrogen instantaneously.The same leaf position was sampled to measure the fresh weight to calculate specific leaf weight.All leaf samples were cut in situ and immediately transferred into a prefrozen 2 mL EP tube, then stored in liquid nitrogen for metabolite extraction.After grinding, each sample was fully dissolved with 800 mL extraction buffer (methanol: chloroform = 7:3 (v/v), -20 °C precooling) and incubated under -20°C for 3 hours.Then 560 mL distilled water (ddH 2 O) was added and mixed with each sample, and 800 mL supernatant was extracted after centrifugation (×2200g, 10min, 4°C).After that, 800 mL buffer (methanol: ddH 2 O = 1: 1(v/ v), 4 °C pre-cooling) was mixed with the sample for another extraction.For each sample, 1.6 mL supernatant in total was obtained by filtering the extraction buffer with a 0.2 mM nylon filter.Among them, 1 mL was used for MS/MS analysis, and 20 mL was used for the QC sample.All extraction operations were performed on ice.

Real-time RT-qPCR
The RT-qPCR analysis was conducted as described previously (Chen et al., 2020).The RNA was extracted with the same procedure as described in Gene expression analysis.For reverse transcription, 0.2 mg RNA was reverse transcribed with TranScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech, China), then SYBR Green Real-Time PCR Master Mix (Yeasen, China) was used for qPCR on the CFX 96 system (Bio-Rad) following the manufacturer's protocol.The ACTIN 8 (AT1G49240) gene was used as a reference for mRNA normalization.The comparative cycle threshold (Ct) method was used to evaluate the relative gene expression levels.The primers used for the expression analysis are listed in Supplementary Table S1.

Measurement of chlorophyll content
Leaf discs (0.8 cm 2 ) were taken from fully expanded leaves, and chlorophyll was extracted with 80% acetone at 4°C for 24 h in darkness, then the supernatant was used for the absorbance measurement at 652 nm in a spectrophotometer (Arnon, 1949).The total chlorophyll content was calculated with the following formula:

Results
The genes related in primary metabolism were induced under low CO 2 condition To study the effect of low CO 2 condition on C 3 plants, we examined the physiological, transcriptomic, and metabolomic changes of Arabidopsis under two CO 2 conditions: low CO 2 at a concentration of 100 ppm (LC) and normal CO 2 at a concentration of 400 ppm (NC).Plants grown under low CO 2 conditions had lower biomass and decreased chlorophyll than those grown under normal CO 2 (Supplementary Figures S1A, B).
The differentially expressed genes (DEGs) were identified between low CO 2 and normal CO 2 treatments (Supplementary Dataset S1).2976 genes were upregulated and 2892 genes were downregulated (Supplementary Figure S2A).Gene Ontology (GO) enrichment analysis shows that hexose catabolic process, sugarphosphatase activity, carbohydrate phosphatase activity, amyloplast, carotene biosynthetic and abscisic acid metabolic process were significantly enriched in upregulated genes (Supplementary Figure S2B; Supplementary Dataset S2), whereas sulfur compound and jasmonic acid-mediated signaling pathway were enriched in the downregulated genes (Supplementary Dataset S2).
In addition, the significantly upregulated genes were mainly participated in metabolic process, including biosynthesis of secondary metabolites, glycolysis/gluconeogenesis, starch and sucrose and carbon metabolism by KEGG analysis (Supplementary Figure S2C; Supplementary Dataset S2).These results show that low CO 2 induced changes to gene expression involved in primary metabolism.
We validated 12 genes that are involved in primary metabolism through RT-qPCR.The results of the RT-qPCR were consistent with the RNA-seq analysis as shown in Supplementary Figure S3A, with a Pearson correlation coefficient of 0.75 (Supplementary Figure S3B).
For ammonium refixation pathway, the gene coding Fd-GOGAT (AT5G04140) also showed significantly higher expression level under low CO 2 condition (Figure 2C).DIT1 and DIT2.1 work in concert in photorespiratory carbon/nitrogen metabolism; the lack of either DiT2.1 or DiT1 led to growth impairment under photorespiratory condition (Kinoshita et al., 2011).In our data, both DiT2.1 and DitT1 showed greater expression levels under low CO 2 (Figure 2C).Moreover, the gene expression of AspAT and AlaAT, which are involved in the transamination, showed significantly increase (Figure 2C).The concentration of 2-oxoglutarate (2-OG) showed no significant change (Figure 2C), whereas the concentration of glutamate was reduced by half (Figure 2C).The concentration of oxaloacetate (OAA) and pyruvate were not changed under low CO 2 condition, while those of aspartate and alanine were slightly decreased (Figure 2C).
Under low CO 2 condition, the expression of the gene AMOS1 (Ammonium-overly sensitive 1) (Li et al., 2012), encoding a plastid metalloprotease that confers sensitivity to ammonium, significantly increased as depicted in Figure 2D.Conversely, the expression of AMOT1 (Ammonium tolerance 1) (Li et al., 2019), which is crucial for mitigating the effects of ammonium toxicity and confers greater tolerance to NH 4 + in mutants than the wild type, was markedly reduced (Figure 2D).All these data suggest that Arabidopsis under low CO 2 was subjected to ammonium stress under low CO 2 condition.

mMDH1 (Figure
The expression levels of genes associated with the glycolysis pathway showed similar trends of changes as those observed among the TCA cycle genes.Notably, most of these genes demonstrated significant upregulation while metabolites like sucrose, glucose-6 phosphate (G6P), fructose-6-phosphate (F6P), fructose-1:6-Bisphosphate (FBP), and triose phosphate (T3P) were significantly reduced under low CO 2 treatment (Figure 3B).We observed increased expression levels of enolase (LOS2, AT2G36530), pyruvate kinase family protein (AT5G08570; AT2G36580), and pyruvate dehydrogenase (PDH-E1 BETA, AT1G30120; PDH-E1_ALPHA, AT1G01090).Among the genes related to the Calvin-Benson-Bassham (CBB) cycle, most of the genes showed upregulation in terms of expression, and the majority of the metabolites were affected (Figure 3C).While pentose phosphate (PenP), FBP, and T3P metabolites decreased significantly, other metabolites mostly showed an increase in their levels under the low CO 2 condition (Figure 3C).
The 2-PG showed the strongest increase compared to other photorespiratory metabolites (Figure 2), which is in well agreement with the transcriptional repression of PGLP1 (Figure 2).Earlier work found that 2-PG inhibits CBB cycle enzymes TPI and SBPase in Arabidopsis after treatment with low CO 2 (Flugel et al., 2017).Such an inhibition may decrease further increase of 2-PG and hence avoid potential greater damage to the cell under low CO 2 .
These results show that under low CO 2 condition Arabidopsis changed expression of primary carbon metabolism and adjusted their metabolite to boost its adaptability to low CO 2 condition.

NH 4 + treatment increased the expression of genes involved in ammonium refixation
In our results, the leaf NH 4 + content increased (Figure 2B) and the ammonium refixation was induced (Figure 2C) following low CO 2 treatment.Plants treated with 30 mM NH 4 + increased the leaf NH 4 + content by at least one-fold, and the NH 4 + content increased by at least three-fold with the 60mM NH 4 + treatment (Figure 4A).
RT-qPCR analysis revealed that NH 4 + treatment upregulated the expression of genes involved in ammonium refixation, specifically GS2 and Fd-GOGAT as shown in Figure 4B.Interestingly, this increase was not observed when plants were treated with NH 4 CI, K 2 SO 4 , KNO 3 , or Mannitol (Li et al., 2019) (Figure 4C).

Comparison of transcriptomics data between NH 4 + treatment and low CO 2 treatment
After NH 4 + treatments, a total of 3920 genes were identified as differentially expressed with 1910 genes upregulated and 2010 genes The effect of low CO 2 on gene expression and metabolites condition in carbon metabolism pathways.downregulated (Supplementary Figure S4A).The GO terms for upregulated genes were enriched in stress, nitrogen component metabolism, carbohydrate biosynthetic process and oxoacid metabolism (Supplementary Figure S4B), which is in line with earlier finding that excessive NH 4 + induced ammonium refixation related genes, such as glutamate dehydrogenase (Patterson et al., 2010;Daniela et al, 2016), and GS/GOGAT pathway (Liu and von Wiren, 2017).The upregulated genes were also significantly enriched in carbon metabolism and 2-oxocarboxylic acid metabolism (Supplementary Dataset S2), with latter term including pyruvate, oxaloacetate and 2-OG which can enter the TCA cycle to generate carbon skeleton for ammonium refixation.We compared the transcriptomic changes induced by both NH 4 + and low CO 2 treatments.We found that DEGs (P<0.05)under both treatments showed a higher Pearson correlation coefficient than those with no significant changes to either treatment (Figure 5A) (P<0.001), which suggest that the treatment of NH 4 + and low CO 2 caused similar changes.The Venn diagram shows that 525 genes were upregulated under both treatments (Figure 5B; Supplementary Dataset S3), while 464 genes were downregulated under both treatments (Supplementary Figure S4C).The jointly upregulated genes are mostly enriched in glycolysis, starch and sucrose metabolism, carbon metabolism, accounting for 36% of those differentially expressed upregulated genes (Figure 5C).The jointly upregulated genes were also found to be enriched in biological processes related to light stimulation, water deprivation, cold, abscisic acid, and the glycolytic process (Figure 5D).These results suggest that multiple genes involved in primary metabolism were simultaneously affected by both NH 4 + and low CO 2 treatments.

Discussion
The gene expression involved in the primary metabolism is adjusted to increase the capacity of ammonium refixation under low CO 2 condition Arabidopsis grown under low CO 2 condition showed a significant decrease in growth (Li Y. et al., 2014) and chlorophyll content compared to the control (Supplementary Figures S1A, B).The stunted growth of Arabidopsis under low CO 2 condition can be attributed to the decreased photosynthetic CO 2 fixation.The observed decrease in chlorophyll concentration may be related to nitrogen deficiency, as chlorophyll concentration depends on nitrogen content (Ercoli et al., 1993).The high photorespiration under low CO 2 leads to an excessive amount of ammonium release in the leaves, which might result in decreased nitrogen content and chlorophyll concentration.Carbon starvation also reduces the capacity of plants to assimilate nitrogen, leading to significant reductions in leaf nitrogen and Rubisco capacity (Dippery et al., 1995;Sage and Coleman, 2001), disrupted C/N ratio (Tcherkez et al., 2017) and induction of autophagic responses (McLoughlin et al., 2020).In our study, carbon starvation resulted in decreased ).Detailed gene information can be found in Supplementary Dataset S3. (C) KEGG enrichment analysis of significantly upregulated genes.(D) Gene ontology (GO) Biological process (BP) enrichment analysis of significantly upregulated genes.The size of the circles represents the enriched gene number, with a larger circle indicating a higher number of genes in the corresponding metabolic pathway category.The horizontal axis represents the P-value.Top 10 significantly enriched results are shown here (C, D).Remaining enriched results can be found in Supplementary Dataset S3.Fisher's exact text P<0.05.
sucrose levels in leaves (Figure 3C).Interestingly, the expression of genes related to amino acid biosynthesis, carbohydrate biosynthesis, carotenoid biosynthesis etc. were increased (Figure 5C).This might be a compensatory effect, which has also been observed in studies of carbon-starved maize (McLoughlin et al., 2020).
The upregulation of PEPC and PEPE-K genes in response to low CO 2 has also been observed in our earlier study (Li Y. et al., 2014).In this new study, we found that the expression of almost all core C 4 cycle enzymes and transporters were upregulated under low CO 2 treatment (Figure 1).Interestingly, a similar upregulation of C 4 like pathway genes for carbon fixation was observed in Nannochloropsis oceanica under low CO 2 condition (Wei et al., 2019).These data suggest a role of low CO 2 condition in the strengthening of C 4 gene expression during the Oligocene period when C 4 emerged genes during evolution.
Low CO 2 condition, like other stresses such as drought and high temperature (Carmo-Silva et al., 2008;Peterhansel and Maurino, 2011), increases photorespiration (Figure 2A) and promotes NH 4 + production (Figure 2B) (Frantz et al., 1982;Alencar et al., 2019).Photorespiration is the primary source of NH 4 + in higher plants (Leegood et al., 1995;Kumagai et al., 2011).Arabidopsis, being an NH 4 + sensitive plant (Cruz et al., 2006), showed stunted plant growth and leaf chlorosis under low CO 2 (Supplementary Figures S1A, B) (Li et al., 2012;Li Y. et al., 2014;Esteban et al., 2016).Taken together, these results suggest that Arabidopsis experiences ammonium stress under low CO 2 condition.Previous studies have shown that excess ammonium is toxic (Hachiya et al., 2012;Li B. et al., 2014;Esteban et al., 2016) and needs to be refixed by upregulation of several key enzymes (Cruz et al., 2006;Liu and von Wiren, 2017;Hachiya et al., 2021).Indeed, the GS/ GOGAT pathway which is involved in ammonium refixation was enhanced under low CO 2 condition (Figure 2C).The TCA cycle under illumination helps provide the carbon skeleton, in particular 2-OG, for ammonium refixation (Nunes-Nesi et al., 2010;Sweetlove et al., 2010;Tcherkez et al., 2017).The gene expression of IDH1 and ACO1, which are used for the generation of 2-OG, were also upregulated in the TCA cycle (Figure 3A).Although the concentration of 2-OG remained unchanged (Arrivault et al., 2009), a significant reduction in the concentrations of fumarate and succinate were observed (Figure 3A), implying that there is an increased flux of 2-OG used for ammonium refixation, rather than decarboxylation in the TCA cycle.Consistent with this, studies have also shown that supplementing with succinate and 2-OG can increase ammonium recirculation and alleviate ammonium toxicity (Yuan et al., 2007;Hachiya et al., 2012).
Previous calculations suggest that the stored leaf citrate content available at the start of light period is insufficient to support the amount of 2-OG required for glutamate production (Stitt et al., 2002).The 2-OG can be generated by reactions catalyzed by aminotransferases.In this aspect, PEPC might play a crucial role during the synthesis of additional 2-OG (Turpin, 1994).Potatoes with overexpressed PEPC display increased 2-OG levels in the leaves (Rademacher et al, 2002); PEPC catalyzes the formation of OAA, which is used in transamination to produce 2-OG.In line with this, the genes encoding PEPC, PEPC-K, AspAT, and MDH are found upregulated under low CO 2 condition (Figure 7).In addition, CAs, which are involved in optimizing cytosolic PEPC activity and ensure normal growth under low CO 2 condition (DiMario et al., 2016;Weerasooriya et al., 2022), increased significantly under low CO 2 condition (Figure 1).Similarly, the expression of PPDK-RP and AlaAT were upregulated, which can also be used to produce more pyruvate and carbon skeletons (Figure 7).Besides, the related transporters, such as Dit1, DIT2.1, showed increased expression as well, which might help meet the increased demand of metabolite transport (Gowik et al., 2011) (Figure 7).All these show that under low CO 2 condition, the primary metabolism can be reprogrammed to generate the required carbon skeletons for ammonium refixation.

NH 4
+ accumulation is a possible signal inducing change in the expression of C 4 related genes under low CO 2 in Arabidopsis How could low CO 2 induce the observed changes in transcriptomics?Here we discuss three possibilities.First, the observed change in transcriptomics might be directly caused by the ammonium accumulation.Indeed, the DEGs identified under low CO 2 and NH 4 + treatments show correlations (Figure 5A).
Specially, under both low CO 2 and NH 4 + treatments, TCA cycle and related aminotransferases which provide 2-OG for ammonium refixation were significantly upregulated (Figure 6A; Supplementary Figure S4).Furthermore, the C 4 related genes were induced by both Ammonium accumulation and refixation is a primary mechanism inducing C 4 related gene expression under low CO 2 condition in Arabidopsis.Low CO 2 treatment caused ammonium toxicity in Arabidopsis.Recirculation of ammonium requires reprogramming of primary metabolism reactions that involved in virtually all core C 4 genes.In vitro NH 4 + treatment induces a similar pattern of changes in C 4 related genes and genes involved in primary metabolism.Therefore, we proposed that increased ammonium is a primary mechanism to induce the expression of C 4 related gene under low CO 2 in Arabidopsis.The Enzymes labeled in different colors were indicated as red/blue: upregulated/downregulated under low CO 2 .Rising/descending arrows beside the enzymes indicated upregulated/downregulated in NH 4 + treatment.NADP-ME and NAD-ME pathways were represented in blue and purple lines respectively.Ammonium recycling pathways were shown in green and yellow rectangles; membrane transporters were indicated in hexagons.
low CO 2 and NH 4 + treatments (Test of independence using Fisher test, P<0.05) (Figure 6B).All these suggest that NH 4 + accumulation can be a strong candidate signal inducing the expression of C 4 related genes under low CO 2 in Arabidopsis.However, the detailed mechanism of how ammonium induced these diverse responses is still unknown.Transcriptomic analysis reveals that among the genes activated in response to NH 4 + , 90% can be regulated by AMOS1 (Li et al., 2012).Interestingly, the transcription level of AMOS1 was increased under low CO 2 (Figure 2D), suggesting AMOS1 might be as a potential candidate to induce C 4 related gene expression.
Secondly, low CO 2 causes decreased photosynthesis, which resulted in decreased biomass production (Supplementary Figure S1A) and decreased concentrations of certain metabolites, such as sucrose (Figure 3B).In general, decreased concentration of carbohydrate enhances expression of genes for CO 2 fixation for both C 3 and C 4 plants and also genes related to mobilization of photosynthesis reserve, and export process (Sheen, 1990;Cheng et al., 1992;Koch, 1996;Ruan, 2014).Given this, decreased sugar concentration might also be a potential mechanism underlining the observed reconfiguration of primary metabolism under low CO 2 condition.
Finally, according to (You et al., 2020), ABA and PEPC play crucial roles in enabling Arabidopsis to adapt to low CO 2 condition.Intriguingly, there was a notable increase in the expression of ABA related metabolic processes under low CO 2 treatment (Supplementary Dataset S2) which implies that ABA signaling might have been involve in the observed changed in the transcriptomic under low CO 2 condition as well.

Implication of the existence of regulatory mechanisms inducing ammonium refixation for C 4 evolution
One major question to answer in the field of C 4 photosynthesis evolution is the detailed process through which rewiring of primary metabolism from C 3 to C 2 photosynthesis occurred.C 2 photosynthesis, which is considered as an intermediate stage during C 4 evolution (Bauwe et al., 2010;Heckmann et al., 2013), has been proposed to use ammonium recycling to resolve the issue of ammonium misbalance between BSC and MC (Mallmann et al., 2014).A number of putative pathways to recycle NH 4 + were proposed by (Mallmann et al., 2014).These pathways usually use many, though not all, enzymes of the C 4 cycle.In all these proposed pathways, ammonium is refixed by GS/GOGAT pathway with carbon skeleton provided by a partial C 4 cycle.Several experimental results support this hypothesis.For example, in C 3 -C 4 intermediate Flaveria species, the transcripts GS/GOGAT are upregulated, as also the case for a number of aminotransferases (Mallmann et al., 2014).The question now needs to answer is that, considering that C 4 photosynthesis emerged from C 3 photosynthesis in a relatively short geological time scale, i.e., only within about ~20 million years (Christin et al., 2008), how could C 3 plants rapidly gain such complex regulatory mechanisms regulating ammonium recycling and providing carbon skeleton when the atmospheric CO 2 drops at Oligocene?
Our results here show that the regulatory mechanism inducing ammonium recycling pathway pre-existed in C 3 plants.Under low CO 2 condition or other conditions where the photorespiratory flux and correspondingly the ammonium release flux are high, an innate regulatory mechanism to induce expression of genes involved in ammonium recycling can be activated.This is sensible since conditions causing physiological low CO 2 condition, e.g.drought, high temperature, etc. which causes stomatal closure (Ehleringer et al, 1991;Wilkinson and Davies, 2002;Sage et al., 2018), widely exist, mechanisms to enable capturing the released ammonium will confer competitive advantage for plants.This innate ammonium recycling mechanism can be used to solve the challenge of ammonium misbalance in C 2 species when this is under demand after the re-allocation of GDC from MC to BSC (Mallmann et al., 2014).This finding is in line with many previous studies which demonstrate that C 4 evolution recruited many pre-existing parts and regulatory mechanisms (Aubry et al., 2011;Reyna-Llorens and Hibberd, 2017;Dickinson et al., 2020), which together underlie the parallel independent evolution of C 4 photosynthesis.

Summary and perspective
Low CO 2 is well recognized to be closely related to the evolution of C 4 photosynthesis.Historically, this is mainly discussed from the perspective of low CO 2 acting as a selection pressure.This study and also some earlier study show that leaves under low CO 2 can also induce changes in the expression of many key C 4 genes.Furthermore, here we show that low CO 2 induces large scale rewiring of the primary metabolism to increase the capacity of leaves to assimilate ammonium, with ammonium accumulation being a possible candidate signal causing the observed changes.These innate mechanisms which induce the up-regulation of genes involved in ammonium refixation can be used to rebalance the ammonia residue after the GDC is reallocated from MC to BSC.Therefore, we propose that the mechanism responsible for the induction of genes involved in ammonium refixation represents a major regulatory mechanism, which facilitated the evolution of C 4 photosynthesis.
Here we list some potential caveats of this study and should be the focus of future studies.First, we point out that during the evolution of C 4 photosynthesis, there is a shift in the spatial expression patterns of critical genes involved in C 4 photosynthesis (Reeves et al., 2017;Taniguchi et al., 2021).Whether low CO 2 might have influenced the spatial expression pattern remains to be studied.Second, in this study, when the CO 2 concentration decreased, the CO 2 :O 2 ratio also decreased.Here we do not differentiate whether the observed responses is caused by the decreased CO 2 level, or decreased CO 2 :O 2 ratio.Thirdly, in this study, we show that the ammonium might be a potential signal inducing the observed changes in gene expression.However, under both low CO 2 and high ammonium concentration, there is an increased carbon assimilation.The possibility of carbon deficiency as a signal for the observed changes also needs to be studied later.Fourthly, the signaling pathway underlies the observe changes in the gene expression needs to be elucidated as well.Elucidation of the detailed molecular mechanism will not only help understand how convergent evolution occurred for C 4 photosynthesis, but also help guide future efforts of evolution guided engineering C 4 prototype.

FIGURE 1
FIGURE 1 FIGURE 2 Changes of the gene expression and metabolites concentration on photorespiratory and ammonium recycling pathways under low CO 2 .(A) Photorespiratory pathway.(B) NH 4 + content in the leaf (replications n=4).(C) Ammonium recycling pathway.(D) The changes of expression of ammonium-sensitive genes.AMOS1 (AT5G35220, Ammonium-overly-sensitive 1), AMOT1 (AT3G20770, Ammonium tolerance 1).The relative changes of gene expression and metabolites are shown in log2Fold change in heatmap, which use the same heatmap scale.For gene expression (A, C, D) differentially expressed genes (DEGs) were determined by the R package 'DESeq2' with the adjusted P-value <0.05 (replications n=4).For metabolites (A, C) and free NH 4 + content in the leaf (B) which were determined by two-sided Student's t-test (replications n=5) and * indicates P<0.05.Data are shown as mean ± s.d (B, D).The symbols of genes in these metabolic pathways are from the Arabidopsis Information Resource (TAIR) Database (http://www.arabidopsis.org/),and also can get from Supplementary Dataset S1.
FIGURE 5 Comparison of transcriptomic profiling between NH 4 + and low CO 2 treatments.(A) Pearson correlation analysis of transcriptomics data between NH 4 + and low CO 2 treatment.The black line represents the distribution of Pearson correlations of randomly selected genes (1632) for 1000 times, and the red line represents the Pearson correlation of differentially expressed 1632 genes.A cutoff value for adjusted P-value < 0.05 were used to identify differentially expressed genes.(B) Venn diagrams.The diagram shows the number of genes which are jointly significantly upregulated (adjusted P-value < 0.05) under low CO 2 treatment (FC-CO 2 ) and NH 4 + treatment (FC-NH 4 + FIGURE 6 (A) Heatmap of differentially expressed genes of different metabolic pathways under NH 4 + treatment (replications n=4).Genes from six pathways were shown as labeled in different colors indicated in the "Pathways" legend.(B) Comparative analysis of C 4 related enzymes under low CO 2 treatment (FC-CO 2 ) and NH 4 + treatment (FC-NH 4 +).The fisher's exact text P<0.05.